Nonsegmented negative strand RNA viruses have a unique feature that its template RNA is always enwrapped by a nucleocapsid protein N. The viral polymerase containing L and P proteins could recognize the template during transcription and replication only when the genomic RNA is associated with N. In the previous period, the crystal structure of a N-RNA complex and that of the central domain of P have been solved. Functional inferences derived from the structure are used in formulating the experiments in this proposal. Four specific aims are presented in this proposal: Aim 1. Structure of the P-N-RNA complex. Our initial analysis indicated that there is a significant conformational change in the N-RNA complex when P is attached. The implication may be that P induces a change in the N-RNA template that is required for the recognition by the L containing polymerase. The complex structure will be determined by combination of cryoEM and X-ray crystallography. The EM structure will provide the framework in which the atomic structures of individual P domains and the N-RNA complex could be built. Aim 2. Dissection of P and N interaction. Based on the three dimensional structures of N and P, we have designed a series of experiments to define the interactions of P with N-0 to reveal their functions. The N0-P complex is recruited into the site of replication and N-0 is then assembled into the nascent N-RNA strand. In this aim, N will be trimmed and mutated so it will not encapsidate RNA nor form large oligomers in the presence of P or its fragments. Furthermore, functions of P related to replication versus transcription will be determined with a novel reverse genetic system. Aim 3. Study of the replicase complex formed by L, N and P. As proposed by Banerjee's group, the VSV replicase complex is formed by L, N and P. We have constructed a cell line that co-expresses L, N and P constitutively. To study this replicase complex functionally and structurally, we will purify the complex in sufficient quantities. The stoichiometry of each component will be determined and the complex will be subject to cryoEM studies and crystallization. Functional inferences from the structure of L, N or P will be tested in the novel reverse genetic system. Aim 4. Structure of the M-P-N-RNA complex. The matrix protein M is required for VSV assembly. However, its role in the process is not clearly defined. Extended from our previous P-N coexpression construct, we have generated a construct that co-expresses M, P and N. M was copurified with the P-N-RNA complex. Crystallization and cryoEM studies of this complex are in progress. The result will show how M may interact with the N-RNA complex, and perhaps with P as well.